Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active β2-microglobulin domain

As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2D^d class I MHC molecule (SCβD^dm) which has β₂-microglobulin (β₂m) covalently linked to the amino terminus of a full-length H-2D^d heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2D^d-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SCβD^dm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27° or 37 °C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SCβD^dm can be stably expressed in β₂m-negative cells, showing that the single-chain molecule uses its own β₂m domain to achieve correct folding and surface expression. Furthermore, the β₂m domain of SCβD^dm, unlike transfected free β₂m, does not rescue surface expression of endogenous class I MHC in the β₂m-negative cells. This strict cis activity of the β₂m domain of SCβD^dm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.     

MHC Class I Phenotype and Function of Human β2-Microglobulin Transgenic Murine Lymphocytes

Lymphoid cells from β₂-microglobulin (β₂m) knockout mice transgenic for human (h) β₂m (C57BL/10 mβ₂m^?/hβ₂m⁺) were compared with normal mice for their binding to exogenously added hβ₂m, binding to a H-2D^b peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous hβ₂m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/hβ₂m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous hβ₂m to MHC-I, in particular, to the H-2D^b molecule through an exchange with endogenous mouse β₂m. In contrast to normal H-2D^b molecules, hybrid H-2D^b expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added hβ₂m suggesting that a stable fraction of hybrid H-2D^b molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/hβ₂m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.     

Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in 'Knock-Out' Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants

Beta-2 microglobulin (β₂m) gene ‘knock-out’ mice (C1D) were primed wilh purified H-2K^b and H-2D^b molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2^b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2^b, H-2^d, as well as H-2^k cells by FACS analysis. The MoAbs stained to two β₂m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous β₂m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β₂m suggesting that xenogeneic β₂m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26°C, however, staining was reduced five-fold when RMA-S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β₂m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.     

Surface expression of β2-microglobulin-associated thymus-leukemia antigen is independent of TAP2

Mouse thymus-leukemia antigen (TL), like other major histocompatibility complex (MHC) class I-b antigens, displays signs of a specialized function. It is normally expressed at high levels on immature thymocytes and at moderate levels on gut epithelium and activated mature T cells. A promoter/enhancer region unique among class I genes accounts for this narrow range of tissue distribution. Like most other class I molecules, TL is dependent upon endogenous β2-microglobulin (β2m) for transport to the surface. However, here we show that unlike most other MHC class I molecules, TL is expressed efficiently in the absence of functional transporter associated with antigen processing subunit 2 (TAP2). A putative fourth TL^a gene cloned from A.SL1 cells was expressed in RMA and RMA-S cells. In bulk transformants, TL expression is higher in TAP2^? RMA-S cells than in wild-type RMA cells, and is not elevated by incubation at reduced temperatures or exposure to exogenous β2m. Analysis of immunoprecipitasted molecules by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that TL is processed normally in RMA-S cells and is associated with β2m both intracellularly and at the cell surface. However, TL heavy chains expressed on the cell surface in the absence of TAP2 are cleaved to a predominant 38 kDa fragment, presumably the result of an altered conformation that renders TL more susceptible to proteolysis. These results suggest that while TL may normally acquire TAP2-dependent peptides, this class I-b molecule does not require them for efficient export to, and stable expression at the cell surface.     

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of β2-microglobulin

F₁ hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F₁ hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2^b/d) devoid of β₂-microglobulin (β₂m) are incapable of rejecting β₂m?/? parental C57BL/6 cells (H-2^b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F₁ mice of the H-2^b/d haplotype, requires expression of MHC class I molecules complexed with β₂m.     

The interaction of beta 2-microglobulin (β2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse β2m

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8⁺ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and β₂-microglobulin (β₂m) have been used to examine the assembly of the trimolecular MHC class I/β₂m/peptide complex. Recombinant human β₂m and mouse β₂m² have been generated to compare the binding of the two β₂m to mouse class I. It is frequently assumed that human β₂m binds to mouse class I heavy chain with a much higher affinity than mouse β₂m itself. We find that human β₂m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse β₂m. In addition, we compared the effect of the two β₂m upon peptide binding to mouse class I. The ability of human β₂m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse β₂m only facilitated peptide binding when mouse β₂m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse β₂m to support peptide binding could not be attributed to a reduced affinity of mouse β₂m/MHC class I complexes for peptides or to a reduction in the fraction of mouse β₂m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in β₂m binding. We propose that mouse β₂m interacts with the minor peptide binding (i.e. the “empty”) fraction with a lower affinity than human β₂m does, whereas mouse and human β₂m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse β₂m is less efficient than human β₂m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human β₂m preferentially.     

Major histocompatibility complex class I-binding peptides are recycled to the cell surface after internalization

Cytotoxic T lymphocytes (CTL) recognize target antigens as short, processed peptides bound to major histocompatibility complex class I (MHC-I) heavy and light chains (β₂-microglobuhn; β₂ m).The heavy chain, which comprise the actual peptide binding α-1 and α-2 domains, can exist at the cell surface in different forms, either free, bound to β₂m or as a ternary complex with β₂m and peptides. MHC-I chains are also known to internalize, and recycle to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-I-bound peptides also can recycle is not known. We have investigated this by using both peptide transporter mutant RMA-S cells and EL4 cells loaded with D^b-binding peptides, by two different approaches. First, peptides were covalently linked with galabiose (Galα4Gal) at a position which did not interfere with D^b binding or immunogenicity, and peptide recycling tested with Gal₂-specific monoclonal antibodies. By flow cytometry, a return of Gal₂ epitopes to the cell surface was found, after cellular internalization and cell surface clearance by pronase treatment. This peptide recycling could be discriminated from free fluid-phase uptake and was inhibited by methylamine, chloroquine and low temperature (18°C) but not by leupeptin. Second, specific CTL were reacted with peptide-loaded target cells after complete removal of surface D^b molecules by pronase, and after different times of incubation at 37C to allow reexpression. By this procedure, reappearance of target cell susceptibility was confirmed. The results are in agreement with a model for optimizing peptide presentation by recycling through an intracellular compartment similar to early endosomes in certain antigen-presenting cells.     

Expression of β2m-Free HLA Class I Heavy Chains in Neuroblastoma Cell Lines

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with β₂-microglobulin (β₂m) by neuroblastoma (NB) cell lines IMR-32 and LA-N-1. The cells, which express barely detectable amounts of β₂m-free (L31-positive molecules) and β₂m-complexed HLA class I antigens (W6.32- and BBM. I-reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon-γ (rIFN-γ) treatment led IMR-32 and LA-N-1 cells to almost exclusively express β₂m-complexed HLA class I heavy chains. Surface β₂m-free MHC class I molecules displayed a molecular mass of ~45 kDa and did not bind exogenously added β₂m. No changes in the synthesis of either HLA class I and β₂m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post-translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of β₂m-free class I heavy chains is a feature of other cell types, such as NB cells.     

Induction and functional characterization of β 2-microglobulin (β2-μ)-free HLA class I heavy chains expressed by β2-μ-deficient human FO-1 melanoma cells

The frequent loss of β2-microglobulin (β₂-μ) in malignant cells has stimulated interest in the functional characteristics of β₂-μ-free HLA class I heavy chains, since this information contributes to assess the impact of β₂-μ abnormalities on the interaction of malignant cells with immune cells. Therefore, the present study has investigated the ability of β₂-μ-free HLA class I heavy chains to modulate NK cell-mediated lysis of melanoma cells and to present melanoma-associated antigen (MAA)-derived peptides to HLA class I-restricted, MAA-specific cytotoxic T lymphocytes (CTL). β₂-μ-free HLA class I heavy chains were induced on B₂m null FO-1 cells by sequential incubation with IFN-α for 48 h at 37 °C and for 24 h at 26 °C. Transfection of cells with a wild-type H-2L^d gene (FO-1L^d) enhanced the induction of β₂-μ-free HLA class I heavy chains under such experimental conditions. β₂-μ-free HLA class I heavy chains expressed on the cell membrane did not protect the B₂m null FO-1 cells from NK cell-mediated lysis. Furthermore, FO-1 cells which express β₂-μ-free HLA-A2 heavy chains following transfection with a wild-type HLA-A2 gene were not lysed by HLA-A2-restricted, MAA-specific CTL lines and clones. These results indicate that association with β₂-μ is required for interaction of HLA class I molecules with NK inhibitory receptors and for peptide presentation to CTL.     

Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous β2- microglobulin

Processing of exogenous hepatitis B surface antigen (HBsAg) particles in an endolysosomal compartment generates peptides that bind to the major histocompatibility complex (MHC) class I molecule L^d and are presented to CD8⁺ cytotoxic T lymphocytes. Surface-associated ‘empty’ MHC class I molecules associated neither with peptide, nor with β2-microglobulin (β2m) are involved in this alternative processing pathway of exogenous antigen for MHC class I-restricted peptide presentation. Here, we demonstrate that internalization of exogenous β2m is required for endolysosomal generation of presentation-competent, trimeric L^d molecules in cells pulsed with exogenous HBsAg. These data point to a role of endocytosed exogenous β2m in the endolysosomal assembly of MHC class I molecules that present peptides from endosomally processed, exogenous antigen.     

The assembly of H2-Kb class I molecules translated in vitro requires oxidized glutathione and peptide

Association of the mouse major histocompatibility complex (MHC) class I heavy chain H2-K^b with mouse β₂-microglobulin (β₂m) was studied in an in vitro translation system. Formation of stable class I complexes was found to be dependent on the presence of presentable peptides and oxidized glutathione, which promotes the formation of disulfide bridges. Translocation of peptides into microsomes was demonstrated by showing that a radioiodinated peptide containing an N-glycosylation acceptor site became glycosylated. Class I complex formation was observed only when heavy chains and β₂m were translated simultaneously, and thus occurs in the microsomes and not after their solubilization. However, peptide binding takes place only after solubilization of the microsomes. The class I complexes translated in vitro show the same specificity and length preference for peptides as their counterparts in RMA-S cells. Assembly of in vitro translated class I complexes was found to occur also in the absence of peptides, resulting in the formation of unstable molecules that are stabilized by incubation with peptides.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Reduced expression of major histocompatibility complex class I free heavy chains and enhanced sensitivity to natural killer cells after incubation of human lymphoid lines with β2-microglobulin

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface - for example as “empty” β₂-microglobulin (β₂m)-associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with β₂m. This was found to shift the equilibrium between β₂m-associated and nonassociated - heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I-deficient cell lines was not affected by β₂m, arguing against a general nonspecific effect of fern on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of β₂m) rather than with expression of complexes.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

β2 -Microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition,but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking β2 -microglobulin (β₂ m− mice) express greatly reduced levels of MHC class I molecules, and cells from β₂ m− mice are therefore highly sensitive NK cells. However, NK cells from β₂ m− mice fail to kill β₂ m− normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of β₂ m− NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to β₂ m− NK cells, we made the following observations: First, β₂ m− NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to β₂ m− concanavalin A (Con A)-activated T cell blasts. Secondly, β₂ m− NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than β₂ m+ NK cells. Third β₂ m− NK cells were not only tolerant to β₂ m− Con A blasts but also to Con A blasts from H-2Kb − /Db − double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of β₂ m− NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in β₂ m− mice, which may instead operate through mechanisms involving triggering pathways.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.     

Lateral organization of the ICAM-1 molecule at the surface of human lymphoblasts: A possible model for its co-distribution with the IL-2 receptor,class I and class II HLA molecules

Lateral distribution of the ICAM-1 molecule and its topological relationship (mutual proximity) to the heavy and light chains of class I HLA molecules, HLA-DR and interleukin-2 receptor α-chain (IL-2Rα) were studied in the plasma membrane of HUT-102B2 T and JY B lymphoblastoid cell lines by the technique of flow cytometric energy transfer (FCET). Effects of adherency and treatments with recombinant interferon-γ or tumor necrosis factor-α on the relative expression level of ICAM-1 to the above cell surface proteins were also investigated. While the cytokines did not significantly affect the ICAM-1 level of either cell line, an increased ICAM-1 expression was found on adherent JY cells. The ICAM-1 expression varied significantly with the cell cycle and culture conditions, as well. The statistical analysis of the differences observed in the energy transfer efficiency histograms resulted in a possible model of lateral co-distribution of these proteins in the plasma membrane. These two-dimensional patterns proved to be different for T and B lymphoma lines. ICAM-1 molecules showed a high degree of self-ssociation on HUT-102B2 (T) cells, while they were mainly expressed as monomers on the surface of JY (B) cells. Both cells showed a significant (ca. 30 %) difference between densities of the heavy and light chains of class I HLA antigen, suggesting a substantial amount of β2-microglobulin free heavy chains on these cell lines. The class I HLA molecules also showed partial self-association, but on both cell lines. The β₂-microglobulin and the heavy chain of the class I HLA showed strongly different proximities to the IL-2Rα, HLA-DR and ICAM-1 molecules, indicating that their orientations relative to the other proteins are dissimilar. IL-2Rα molecules of the HUT-102B2 (T) cells are located mostly in the vicinity of the β₂-microglobulin. In contrast, the local density of HLA-DR antigens is higher in the proximity of the heavy chain than in the vicinity of the β₂-microglobulin. The possible functional significance of these protein patterns is also discussed herein.